Benzilylcholine mustard and spare receptors in guinea pig ileum

A comparison was made of muscarinic receptor occupancy by the irreversible antagonist benzilylcholine mustard (BCM) as determined from shifts in the dose-response curve to a muscarinic agonist and from 3H-QNB binding to homogenates of BCM-treated tissue. Major discrepancies were found. A low concentration of BCM (3x10-8M/15 min.) produced a parallel dose-response curve shift corresponding to 98-99% receptor occupancy by BCM, whereas 3H-QNB binding revealed only 48% receptor occupancy. Possible origins of this discrepancy are discussed. High concentrations of BCM (5x10-5M, 15 min.) fail to completely alkylate all 3H-QNB binding sites even though response is completely lost. Although significant (64%) recovery of response occurs after prolonged tissue washing (240 min.) this is not accompanied by an increase in 3H-QNB binding. The small fraction (approximately 5%) of sites inaccessible to BCM and with reduced affinity for 3H-QNB may represent a subpopulation of muscarinic receptors.